High-throughput screening method of hepatic stellate cell activation inhibitor and application thereof

A hepatic stellate cell and screening method technology, which is applied in the field of high-throughput screening of hepatic stellate cell activation inhibitors, can solve the problems of small Stokes shift, difficult high-throughput screening method, cumbersome process, etc. easily measurable effect

Pending Publication Date: 2021-05-11
XIAMEN BERYL THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the problems existing in the application of BODIPY are that the Stokes shift is small and the photostability is not good.
In one case, ORO staining was used to mark that HSC activation was inhibited by inhibitors, but based on light microscope observation, photography, image analysis and quantification, the process is cumbersome and it is not easy to form a high-throughput screening method
At present, there is no report on high-throughput screening of HSC activation inhibitors using microplate reader for fluorescence quantification

Method used

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  • High-throughput screening method of hepatic stellate cell activation inhibitor and application thereof
  • High-throughput screening method of hepatic stellate cell activation inhibitor and application thereof
  • High-throughput screening method of hepatic stellate cell activation inhibitor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] A high-throughput screening method for hepatic stellate cell activation inhibitors, comprising the following steps:

[0048] Step 1: Using a combination of sodium oleate, vitamin A, and insulin to restore hepatic stellate cells to a resting state;

[0049] Step 2: Use fetal bovine serum, TGF-β1, PDGFBB / AA, lipopolysaccharide (LPS) or any combination thereof to induce reactivation of hepatic stellate cells; at the same time, add compounds to be screened to observe the inhibitory effect of a certain molecule on the induction and activation conditions ;

[0050] Step 3: Stain the hepatic stellate cells with a dye, read the fluorescence or light absorbance with a microplate reader for quantitative analysis; screen the drug to be tested, select the candidate active drug according to the activity, and confirm the drug by Western blot or RT-PCR method effect. The specific method steps are as follows:

[0051] Oil red O-methylene blue double staining method (ORO-MB method) i...

Embodiment 2

[0055] Using Bodipy / -Hoechst33342 double-stained fluorescence method (B-H method) for lipid titer in cells:

[0056] In the previous example, 4% neutral paraformaldehyde was fixed at 4 degrees Celsius for 12 hours, discarded the fixative and dried it completely, washed twice with PBS, and 50 μl of fluorescent staining solution (1 μg / ml Bodipy493 / 503 in PBS, B and 0.5 μg / ml ml Hoechst33342, H) 37°C for 20 minutes in the dark, discard the dye solution, wash 3 times with PBS, read H-350 / 461nm and B-493 / 503nM on a microplate reader, and calculate the B / H fluorescence intensity.

[0057] Considering that there are fluorescent chromophores in the structure of some compounds, autofluorescence around 490-510nm will affect the screening and cause false positives, so Bodipy with other emission wavelengths, that is, more than two BODIPY series compounds with different excitation / emission wavelengths, are used as probes. Needle, multi-channel fluorescent screening to avoid false positives...

Embodiment 3

[0060] Lipid titer detection and verification of known drugs that inhibit HSC activation:

[0061]Some small molecule drugs that can inhibit the activation of hepatic stellate cells in the literature include rosiglitazone (CAS 122320-73-4), rutin (Rutin, CAS 153-18-4), chloroquine (Chloroquine, CQ, CAS 54-05-7), curcumin (Curcumin, CAS 458-37-7) and L-732138 (CAS 148451-96-1) were used as positive drugs, which were detected by the aforementioned ORO-MB staining and quantitative methods The inhibitory effect of positive drugs on the activation of LX-2 cells induced by TGF-β1, LPS, PDGFAA / BB and FBS is manifested as an increase in the amount of lipid droplets, and the corresponding EC50 measured is shown in Figure 4-Figure 9.

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Abstract

The invention discloses a high-throughput screening method of a hepatic stellate cell activation inhibitor. The high-throughput screening method comprises the following steps of: step 1, restoring hepatic stellate cells to a resting state by using a combination of sodium oleate, vitamin A and rosiglitazone or a combination of sodium oleate, vitamin A and insulin; step 2, jointly inducing the hepatic stellate cells to be activated again by using fetal calf serum, TGF-beta 1, PDGFBB / AA, lipopolysaccharide or any combination thereof respectively; and meanwhile, adding a compound to be screened to observe the inhibition effect of a certain molecule on an induced activation condition; and step 3, dyeing the hepatic stellate cells by using a dye, reading fluorescence or light absorption amount by using a microplate reader for quantitative analysis; screening a drug to be detected, selecting a candidate active drug according to activity, and verifying the drug effect. The invention also discloses application of the method in quantitative analysis of the drug effect of a targeted drug for determining hepatic stellate cell activation inhibition. The high-throughput screening method disclosed by the invention can be used for quantitatively detecting the activation state of the hepatic stellate cells.

Description

technical field [0001] The invention relates to the field of biochemistry, in particular to a high-throughput screening method for hepatic stellate cell activation inhibitors and its application. Background technique [0002] The activation of hepatic stellate cells is a key link in the process of liver fibrosis, and there is currently no targeted drug that can inhibit the activation of hepatic stellate cells. The lack of quantitative detection methods for the activation state of hepatic stellate cells also makes it difficult to measure the efficacy of inhibitors. [0003] 1. Liver fibrosis and hepatic stellate cell activation [0004] Liver fibrosis is a wound repair response of the body, and the activation of HSC in the damaged area of ​​liver cells is surrounded by a large amount of extracellular matrix, which provides a potential target for anti-fibrosis treatment of liver fibrosis. The process of liver fibrosis has gone through: 1) liver damage caused by various facto...

Claims

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Application Information

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IPC IPC(8): C12Q1/02G01N21/64
CPCG01N33/5067G01N33/5038G01N21/6486G01N2500/10
Inventor 王阳柏旭
Owner XIAMEN BERYL THERAPEUTICS INC
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